If you are using a purified protein, or an extract containing this protein, and you have some sort of in vitro assay for its activity, then the first thing to do is read the literature to see what others have done as far as optimization. The second thing to do is to vary the conditions of the assay to find out what the optimal conditions are.
Test a range of pHs with set of several buffers with overlappping pH ranges. For example, MOPS at pH 6.5, 7.0 and 7.5; HEPES at pH 7.0, 7.5 and 8.0; Tris at pH 7.5, 8.0, and 8.5. This experiments allows you to identify the optimal pH as well as the optimal buffer salt.
Test a range of salt concentrations using a variety of salts. Examples of cations are ammonium, sodium, and potassium. Examples of anions are acetate, glutamate, chloride, and sulfate. Test concentrations from zero to perhaps 250 mM. This experiment allows you to identify the optimal salts and the optimal ionic strength.
Test the effect of potential stabilizers such as glycerol (up to 20%), sucrose (up to 1 M), and trehalose (up to 0.5 M).
Test the effect of divalent metals, especially Ca2+, Mg2+ and Mn2+ at concentrations up to 10 mM.
If it is a binding interaction that you are measuring, each measurement would ideally consist of a Kd measurement. However, this could be very laborious in some cases, or use too much material. In that case, you could simplify the experiment by measuring the effect of each condition using concentrations of the binding partners that allow only a relatively low amount of binding under the original condition, so that any improvement in binding will be observable.
What factors are contributing to immunogenicity of a Protein?
Here is a set of tools for epitope prediction:
http://tools.iedb.org/main/
Thank you Adam, I found this site and already did some prediction
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